Chapter 6
Resveratrol and Piceid Levels in Wine Production and in Finished Wines 1
1
Rosa M . Lamuela-Raventós , Α. I. Romero-Pérez , Andrew L. Waterhouse , M . Lloret , and M . C. de la Torre-Boronat Downloaded by NORTH CAROLINA STATE UNIV on December 18, 2012 | http://pubs.acs.org Publication Date: March 1, 1997 | doi: 10.1021/bk-1997-0661.ch006
2
1
1
1
Nutriciói Bromatologia, Facultat de Farmàcia, Universidad de Barcelona, Avinguda Joan ΧΧIII s/n, 08028-Barcelona, Spain Department of Viticulture and Enology, University of California, Davis, CA 95616-8749
2
A direct HPLC method has been developed for the quantification of four resveratrol derivatives, the cis and trans isomers of resveratrol and their glucosides, the piceids. The concentration of these compounds was determined in 60 samples of red, rosé and white wine. Analysis of the low levels in the white wines required an additional sample concentration step. This method was also applied to following the levels of these four compounds during the fermentation and maceration, at a winery scale, of one Merlot and two Cabernet Sauvignon wines. The presence or absence of glycosylation affected the extraction rate while a comparison of the three fermentations showed that each compound had a characteristic extraction behavior.
Heart disease is the first leading cause of death in developed countries. A healthy diet is one of the factors that can contribute to reduce cardiac disease mortality. The traditional Mediterranean diet is accepted as a relatively healthy diet and it has been promoted by the Harvard School of Public Health, United Nations World Health Organization/Food and Agriculture Organization (WHO/FAO), and Oldways Preservation & Exchange Trust. One of the components included in this diet is wine, in moderation, normally with meals. Epidemiological studies show that wine consumption is related to a decrease in cardiovascular disease (1) (2); however, these beneficial effects are not observed among beer and spirits drinkers (2). It has been hypothesized that the compounds responsible for this positive effect are the phenols present in wine (3). The presence of the phenols,frvms-resveratroland its glucoside, piceid, (see Figure 1) in wines has been of much interest for their physiological properties. /raws-Resveratrol is one of the compounds present in wines that could be responsible for the decrease in coronary heart disease observed among wine drinkers, since it
© 1997 American Chemical Society
In Wine; Watkins, T.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
Downloaded by NORTH CAROLINA STATE UNIV on December 18, 2012 | http://pubs.acs.org Publication Date: March 1, 1997 | doi: 10.1021/bk-1997-0661.ch006
6. LAMUELA-RAVENTOS ET AL.
Resveratrol & Piceid Levels in Wine
57
inhibits L D L oxidation (4), it blocks platelet aggregation (5); (6) and eicosanoid synthesis (6, 7). Piceid, the 3-P-glucoside of tams-resveratrol, also shows antiplatelet aggregation properties (6, 7), and it may releasefrvms-resveratrolby the β-glucosidase hydrolysis, after ingestion (8). However, the levels of these compounds required to inhibit platelet aggregation are not very clear, since big differences are found among the antiaggregation reports. Pace-Asciak et al. (1995) (6) reported that 129.9±64.4 μπιοΙ/L of /ra/w-resveratrol can inhibit platelet aggregation by 50%, when ADP is used as inducer, or 164.7±67.3 μιηοΙ/L, when thrombin is the inducer; while Bertelli et al (1995) (5) found that 0.016 μιηοΙ/L inhibited platelet aggregation by 50.3%, when collagen was the inducer. Similar disparities are reported with the inhibition of eicosanoid synthesis; the levels which have physiological effects by Pace-Asciak et al (1995) (6) are 10 times higher than those reported by Kimura et al (1985) (7). The activity of the cis isomers in L D L oxidation. The cis isomers show that they also have, as the trans isomers, potential anticancer activity by inhibiting protein-tyrosine kinase (9). In grape berries, resveratrol synthesis is primarily located at the skin cells and it is absent or low, in the fruit flesh. In red vinification, maceration with skins and seeds, during fermentation, contribute to the extraction of the phenols present in the firmer tissue. Resveratrol requires relatively long maceration time on the skins to be extracted (10-13). The extraction of resveratrol in fermentation is controlled by the increase in ethanol concentration, the enzymatic activity (14) and presumably phenols are affected by absorptive interaction with yeast (15, 16), which settle out after fermentation, and by the fermentation temperature. Red wines have the highest frvms-resveratrol content, since they are macerated with skins for a longer time (13, 17-20). In the only study done in rosé wines (20), /rajw-resveratrol levels were between 0.005 to 1.19 mg/L, with higher amounts in the wine related to longer maceration.White wine is reported to have much lower resveratrol content than the red wine (19, 21, 22), presumably due to minimal skin contact associated with white wine production. Jeandet et al. (1995) (19) noted that white wines macerated with skins had a higher resveratrol content, than nonmacerated ones. Goldberg et al. (1993) (23) describe the minimum and a maximum levels for white wines, and they note that one Sauvignon blanc had the highest level, 1.3 mg/L, while the levels in other white wines were so low that they could not be quantified. Since 1992, when Siemann and Creasy (17) described the presence of the phytoalexin /rajw-resveratrol in wines, many different methods have been described to determine this compound in wines, including these based on HPLC with U V detection (18, 22, 24, 25), and by GC/MS (19, 23, 26, 27), these method required prior sample treatment, before injection. In 1994, McMurtrey et al,. (21) and Pezet et al (22) developed a direct HPLC analysis using electrochemical and fluorimetric detectors, respectively. These methods were very sensitive and rapid. However, they only analyzed frvms-resveratrol. The HPLC method developed by Lamuela-Raventos et al (28) allowed the determination of the four resveratrol derivatives in red, rosé and white wines; however, in white wines a concentration step was necessary to quantify the four resveratrol isomers. Since two reports have claimed that frvms-resveratrol is unstable to rotary
In Wine; Watkins, T.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
58
WINE: NUTRITIONAL AND THERAPEUTIC BENEFITS
evaporation (22, 24), and this technique is used in the analysis of white wines, the stability of these compounds in the rotovap was confirmed. We report the levels of the four resveratrol derivatives in red, rosé and white Spanish wines and we also follow the evolution pattern of the four compounds during fermentation to establish the extraction kinetics of these compounds during fermentation.
Downloaded by NORTH CAROLINA STATE UNIV on December 18, 2012 | http://pubs.acs.org Publication Date: March 1, 1997 | doi: 10.1021/bk-1997-0661.ch006
Material and Methods Samples. Red wines. Wines from different harvests and appellations were analyzed. The varieties tested were Cabernet Sauvignon, Merlot, Pinot noir, Tempranillo, and Grenache. Rosé wines. Three different varieties were tested Cabernet Sauvignon, Pinot noir, and Grenache, being the only varietal rosé wines in the Barcelona market. White wines. The white wine grape varieties analyzed were Albarino, Chardonnay, Macabeo, Parellada, White Riesling, Sauvignon blanc, Verdejo and Xarel.lo. Fermentation curves in red vinification. Cabernet Sauvignon (40,000 Kg) and Merlot (20,000 Kg) were destemmed, crushed, and fermented in the presence of the skins and seeds. Three different tanks of approximately 12,000 L were vinified separately, two of Cabernet Sauvignon and one of Merlot. Samples were collected every two days until the fermentation had finished. Samples of the musts were treated with 1 g/L of NaF to stop the fermentation, and frozen at - 21 °C. Samples were centrifuged (1800g χ 20 min.). Absorbance at 280 nm for total phenols and 320 nm for hydroxycinnamates were determined in these samples, in 1 mm and 10 mm cells, respectively, in a Hewlett-Packard 8452A diode array spectrophotometer, according to the method described by Somers and Ziemelis (1985) (29). Total anthoçyanins were measured following Somers and Evans (1977) (30). Stilbenes determination. Red and rosé wine samples were analyzed by direct HPLC injection, after filtration through Whatman inorganic Anopore membrane filters (Anodisc. 0.2 μιη).Quantitation of the low levels of the cis forms in some white wines required an additional sample concentration step; 10 mL was concentrated to 1 mL by rotary evaporation (30°C, in vacuo) and the concentrate was filtered through a Whatman inorganic Anopore membrane filter, with a prefilter of glass microfiber to avoid plugging the membrane filter (Anotop 10 PlusTM, 0.2μπι). The precision, recovery and reproducibility, of the method were established following The U . S. Pharmacopoeia (USP ΧΧΠ) (31). All the samples were protected from light to avoid light-induced isomerization. H P L C analysis. A Hewlett-Packard (HP) 1050 instrument equipped with a Rheodyne injection valve (Model 7125), ( 100 μL fixed loop) and a diode-array UV-vis detector HP 1040 M coupled to a Chem Station HP 79995A. The column used was a Tracer Nucleosil, C18 120 (25 cm χ 0.4 cm), 5 μιη particle size, with a precolumn of the same material, maintained at 40 °C. Results of the analyses are expresses in equivalents of irans-resveratrol, based on absorbance of the trans and cis isomers at 306 and 285 ran, respectively.
In Wine; Watkins, T.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
6. LAMUELA-RAVENTOS ET AL.
Resveratrol & Piceid Levels in Wine
59
Results and Discussion
Downloaded by NORTH CAROLINA STATE UNIV on December 18, 2012 | http://pubs.acs.org Publication Date: March 1, 1997 | doi: 10.1021/bk-1997-0661.ch006
The validation of the HPLC method was described previously (28). Red wines. Pinot noir wines have the highest levels of frvms-resveratrol, as was observed previously in California (18, 21) and in French wines (25). However, the total amount of resveratrol derivatives were very similar in Merlot and in Pinot noir (see Figure 2). frvms-Resveratrol was the major compound followed by /rows-piceid. So the trans forms predominate compared to the cis ones. The compound present at lowest concentration was cw-resveratrol. Rosé wines.The levels of resveratrol monomers are between the levels of white and red wines. iraws-Resveratrol levels were similar to those described in the only previous study done on rosé wines (20). On these rosé wines, the content of resveratrol does not depend on variety (see Figure 3), presumably because the time of maceration with the skins depends on the technology applied by each winery. In average, the levels of /rans-resveratrol were very similar to trans-piceid. Rosé wines are made with short maceration time with skins, so /rajw-resveratrol, because of its polarity, is not totally extracted from the skins. Again, c/s-resveratrol is the compound present at the lowest concentration. White wines. The degradation of resveratrol during rotary evaporation, as described by Pezet et al. (22) was not observed, and the amounts of the four compounds were the same before and after concentration. White wines had the lowest resveratrol content (see Figure 4). The variety affected the amount of resveratrol isomers present. The traditional Spanish varieties, Xarel.lo, Parellada, Albarino, Verdejo, and Macabeo, had higher amounts of these compounds than the varieties more recently introduced to Spain, Sauvignon blanc, white Riesling, and Chardonnay, (p
